Remedies or preventives containing cyclopentenone compounds as the active ingredient

ABSTRACT

Remedies or preventives for diseases with a need for immunoregulation, diseases with a need for inhibition of inflammation, diseases with a need for regulation of tumor necrosis factor production, diseases with a need for regulation of fungal growth, diseases with a need for regulation of cell adhesion or disease with a deed for induction of heat-shock protein, which contain as the active ingredient at least one compound selected from among cyclopentenone derivatives represented by general formula [I], optically active isomers and salts thereof, wherein R 1  and R 2  are the same or different and each represents hydrogen, an aliphatic group, an aromatic group or an aromatic aliphatic group.

REFERENCE TO RELATED APPLICATIONS

The present application is the national stage under 35 U.S.C. §371 ofinternational application PCT/JP99/04323, filed Aug. 10, 1999 whichdesignated the United States, and was not published in English.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition containinga cyclopentenone ester as an active ingredient.

BACKGROUND ART

Wide variety of drugs including alkylating agents, antimetabolites,carcinostatics such as vegetable alkaloids, antibiotics, immunoenhancersand immnoregulators are conventionally used for clinical therapies.However, pharmacotherapy using such drugs has not completed yet.

Among these, naturally occurring prostaglandins having α,β-unsaturatedcarbonyls in their five-membered rings, i.e., prostaglandins A and J,were reported to suppress DNA synthesis, suggesting their possible useas highly safe carcinostatics. Various derivatives thereof weresynthesized (see JP-A 62-96438).

OBJECTS OF INVENTION

The main object of the present invention is to develop a cyclopentenonederivative having various physiological activities and to provide apharmaceutical composition containing the compound as an activeingredient.

These and other objects as well as advantages of the present inventionwill be explained below in detail with reference to the attacheddrawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the ¹H-NMR spectrum of diisobutyrylcyclopentenone.

FIG. 2 illustrates the ¹H-NMR spectrum of dimethoxyacetylcyclopentenone.

FIG. 3 illustrates the ¹H-NMR spectrum of dimethylfumarylcyclopentenone.

FIG. 4 illustrates the ¹H-NMR spectrum of dimethylmaleylcyclopentenone.

FIG. 5 illustrates the relationship between the amount of cyclopentenonederivative administered and the rate of increase in edema in foot.

FIG. 6 illustrates the relationship between the amount of cyclopentenonederivative administered and the rate of increase in delayedhypersensitivity.

FIG. 7 illustrates the relationship between the concentration ofdipropionylcyclopentenone and the amount ³H-thymidine uptake.

FIG. 8 illustrates the relationship between the concentration ofdipropionylcyclopentenone and the amount of ³H-thymidine uptake.

SUMMARY OF INVENTION

The present inventors have studied intensively in order to accomplishthe objects and found that a cyclopentenone derivative of formula [I]:

(wherein R₁ and R₂ may be identical or different each other, and arehydrogen, an aliphatic group, an aromatic group or an aromatic aliphaticgroup) is produced by reacting 4,5-dihydroxy-2-cyclopenten-1-one offormula [II] (hereinafter simply referred to as cyclopentenone):

with a carboxylic acid and/or a reactive derivative thereof, and thatthe cyclopentenone derivative has strong physiological activities suchas an immunoregulatory activity, an anti-inflammatory activity, anactivity of inhibiting tumor necrosis factor production, an antifungalactivity and an activity of inhibiting cell adhesion. Thus, the presentinvention has been completed.

Accordingly, the present invention provides a pharmaceutical compositionwhich contains as an active ingredient at least one compound selectedfrom the group consisting of a cyclopentenone derivative of formula [I]or an optical isomer thereof, and a salt thereof, said composition beingused for treating or preventing a disease that requires immunoregulationfor its treatment or prevention, a disease that requires suppression ofinflammation for its treatment or prevention, a disease that requiresinhibition of tumor necrosis factor production for its treatment orprevention, a disease that requires inhibition of a fungus for itstreatment or prevention, a disease that requires inhibition of celladhesion for its treatment or prevention, or a disease that requiresinduction of heat shock protein for its treatment or prevention.

DETAILED DESCRIPTION OF THE INVENTION

Cyclopentenones used in the present invention include isomers havinghydroxyl groups at 4- and 5-positions configured in cis and isomershaving the hydroxyl groups configured in trans. A cis or trans isomer ofcyclopentenone or a mixture of the cis and trans isomers may be used inthe present invention. Optical isomers thereof may also be used.

A cis isomer of cyclopentenone is obtained according to a chemicalsynthesis method [Helvetica Chimica Acta, 55:2838-2844 (1972)]. A transisomer of cyclopentenone is obtained according to a chemical synthesismethod [Carbohydrate Res., 247:217-222 (1993)] or by heating uronic acid(e.g., glucuronic acid), a uronic acid derivative (e.g.,glucronolactone) or the like (see WO 98/13328). These heat treatmentproducts containing cyclopentenone and products partially purified orpurified therefrom can be used in the present invention.

For example, cyclopentenone is produced in a heat treatment product byheating a 1% solution of D-glucuronic acid as uronic acid at 121° C. for4 hours. Cyclopentenone in the heat treatment product is extracted witha solvent. The extract is concentrated. The concentrate is thenseparated on silica gel column chromatography. Eluted fractionscontaining cyclopentenone are concentrated. Cyclopentenone is extractedfrom the concentrate with chloroform. The concentrated extract issubjected to normal phase column chromatography, thereby isolatingcyclopentenone in the heat treatment product.

Optical isomers, (−)-4,5-dihydroxy-2-cyclopenten-1-one and(+)-4,5-dihydroxy-2-cyclopenten-1-one, can be obtained by opticallyresolving the thus isolated cyclopentenone. Of course, synthesizedcyclopentenone can be optically resolved.

A cyclopentenone derivative of formula [I]:

or an optical isomer thereof of the present invention is produced in areaction mixture by simultaneously or sequentially reactingcyclopentenone and/or an optical isomer thereof with a carboxylic acidhaving hydrogen, an aliphatic group, an aromatic group or an aromaticaliphatic group and/or a reactive derivative thereof.

The following carboxylic acids having hydrogen, an aliphatic group, anaromatic group or an aromatic aliphatic group and corresponding to R₁and R₂ in the cyclopentenone derivative of formula [I] or reactivederivatives thereof are used in the present invention.

Formic acid can be used as a carboxylic acid having hydrogen.

A carboxylic acid having an alkyl group and a carboxylic acid having analkenyl group can be used as a carboxylic acid having an aliphaticgroup.

A carboxylic acid having a linear or branched alkyl group can be used asa carboxylic acid having an alkyl group. Although the length of thealkyl chain can be suitably selected depending on the biologicalactivity, solubility or the like of the cyclopentenone derivative, agroup of C1-30 is usually preferable. Examples of carboxylic acidshaving linear alkyl groups which can be used include acetic acid,propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoicacid, n-octanoic acid, pelargonic acid, n-decanoic acid, undecanoicacid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid,palmitic acid, heptadecanoic acid, stearic acid, nonadecanoic acid,icosanoic acid, behenic acid, lignoceric acid, cerotic acid and melissicacid.

Examples of carboxylic acids having branched alkyl groups which can beused include isobutyric acid, isovaleric acid, 2-methylbutyric acid,pivalic acid, 4-methylvaleric acid and 1,2-dimethylvaleric acid.

A carboxylic acid having a linear or branched alkenyl group can be usedas a carboxylic acid having an alkenyl group. Although the chain length,the degree of unsaturation and the position of unsaturated bond of thealkenyl group can be suitably selected depending on the biologicalactivity, solubility or the like of the cyclopentenone derivative, agroup of C2-30 is usually preferable.

Examples of carboxylic acids having linear alkenyl groups which can beused include acrylic acid, vinylacetic acid, crotonic acid, isocrotonicacid, allylacetic acid, 2-hexenoic acid, 3-hexenoic acid, 3-octenoicacid, obtusilic acid, 10-undecenoic acid, palmitoleic acid, petroselinicacid, elaidic acid, oleic acid, linoleic acid, α-linolenic acid,γ-linolenic acid, eleostearic acid, icosatrienoic acid, arachidonicacid, eicosapentaenoic acid, brassidic acid, erucic acid,docosahexaenoic acid, ximenic acid and 21-triacontenoic acid.

Examples of carboxylic acids having branched alkenyl groups which can beused include methacrylic acid, tiglic acid, angelic acid andα-ethylcrotonic acid.

A carboxylic acid having an alkyl group that has a lower alkoxyl groupof C1-4 as a substituent such as methoxyacetic acid can be used as acarboxylic acid having a substituted aliphatic group. A carboxylic acidhaving an alkenyl group that has a lower alkoxycarbonyl of C2-5 as asubstituent such as methylmaleic acid can be used.

Examples of carboxylic acids having aromatic groups which can be usedinclude benzoic acid, toluic acid, chlorobenzoic acid, bromobenzoicacid, nitrobenzoic acid, phthalic acid, isophthalic acid, terephthalicacid, salicylic acid, acetylsalicylic acid, acetylsalicylsalicylic acid,aminosalicylic acid, p-hydroxybenzoic acid, aminobenzoic acid,methoxybenzoic acid, acetamidobenzoic acid, vanillic acid, orsellinicacid, naphthoic acid, cinchomeronic acid, xanthurenic acid, quinic acidand kynureic acid. A carboxylic acid having an aromatic group used maybe selected depending on the biological activity, solubility or the likeof the cyclopentenone derivative to be produced.

Examples of carboxylic acids having aromatic aliphatic groups which canbe used include phenylacetic acid, phenylpropionic acid, phenyllacticacid, phenylpyruvic acid, cinnamic acid, atropic acid and naphthylaceticacid. A carboxylic acid having an aromatic aliphatic group used may beselected depending on the biological activity, solubility or the like ofthe cyclopentenone derivative to be produced.

The aliphatic, aromatic or aromatic aliphatic group may have asubstituent such as a functional group (e.g., an amino group, a nitrogroup, an oxo group, a hydroxyl group, a thiol group or a sulfate group)or a halogen (e.g., flourine, chlorine, bromine or iodine).

Thus, R₁ and R₂ in formula [I] may be identical or different each other,and examples thereof include hydrogen, a linear or branched C1-30 alkylgroup, a linear or branched C2-30 alkenyl group, a C6-10 aryl group anda C1-30 alkyl C6-10 aryl group. They may be optionally substituted withat least one substituent selected from the group consisting of a C1-30alkyl group, a C1-4 alkoxy group, a C2-5 alkoxycarbonyl group, an aminogroup, a nitro group, an oxo group, a hydroxyl group, a thiol group, asulfate group and a halogen (e.g., flourine, chlorine, bromine oriodine).

Reactive derivatives of carboxylic acids are exemplified by an acidhalide, an acid anhydride, an acid ester and a salt. A reactivederivative of the carboxylic acid to be used may be produced dependingon the objects.

The reaction between a carboxylic acid or a reactive derivative thereofand cyclopentenone may be carried out such that R₁ and R₂ in thecyclopentenone derivative become identical or different each other.

Specifically, carboxylic acids having different groups for R₁ and R₂ maybe simultaneously reacted with cyclopentenone. Alternatively, they maybe reacted sequentially. In the latter case, a cyclopentenone derivativein which R₁ and R₂ are different each other can be efficiently producedby protecting one of the hydroxyl groups of cyclopentenone.

A cyclopentenone derivative or an optical isomer thereof produced byreacting cyclopentenone or an optical isomer thereof with carboxylicacid has an immunoregulatory activity, an anti-inflammatory activity, anactivity of inhibiting tumor necrosis factor production, an antifungalactivity, an activity of inhibiting cell adhesion or the like. Thecyclopentenone derivative or an optical isomer thereof can be purifiedand isolated from the reaction mixture using one of these activities asan index. Known purification/isolation means including chemical meansand physical means may be used for purification and isolation.Conventional purification means such as gel filtration, fractionationusing a molecular weight fractionating membrane, solvent extraction,fractional distillation and various chromatographies using, for example,ion-exchange resins can be used in combination to purify and isolate thecyclopentenone derivative or an optical isomer thereof from the reactionproduct.

For example, a cyclopentenone derivative used in the present inventionis produced by dissolving cyclopentenone or an optical isomer thereof,4-dimethylaminopyridine and carboxylic acid in dichloromethane andadding N,N-dicyclohexylcarbodiimide thereto for reaction while coolingon ice. The cyclopentenone derivative of interest can be isolated bypurifying the product on silica gel thin-layer cromatography.

Furthermore, diacetylcyclopentenone can be purified and isolated from areaction product obtained by reacting cyclopentenone or an opticalisomer thereof with acetic anhydride in anhydrous pyridine.

Optical isomers of the cyclopentenone derivatives used in the presentinvention can be separated by mechanical resolution of racemic mixture,preferential crystallization, resolution by crystallizing as adiastereomeric salt or an inclusion compound, kinetic resolution usingan enzyme or a microorganism, chromatographic separation or the like.

Gas chromatography, liquid chromatography, thin-layer chromatography orthe like using an appropriate chiral stationary phase can be used forchromatographic resolution.

A method in which a chiral stationary phase is used, a method in which achiral eluent is used, separation as a diastereomer or the like can beused for optical resolution by liquid chromatography.

An amide-type stationary phase, a urea-type stationary phase, a ligandexchange-type stationary phase, a polysaccharide or polysaccharidederivative stationary phase, a protein stationary phase, apolymethacrylate ester stationary phase, a polymethacrylamide stationaryphase or the like can be used as a chiral stationary phase.

A hexan-type eluent, an alcohol-type eluent, an aqueous (buffer) eluentor the like can be appropriately used as an eluent depending on thestationary phase used.

Salts of the cyclopentenone derivatives or optical isomers thereof usedin the present invention include pharmaceutically acceptable salts.Known methods can be used for the conversion.

Representative examples of the cyclopentenone derivatives used in thepresent invention include diacetylcyclopentenone,dipropionylcyclopentenone, dibutyrylcyclopentenone,diisobutyrylcyclopentenone, divalerylcyclopentenone,dihexanoylcyclopentenone, dioctanoylcyclopentenone,didecanoylcyclopentenone, dimyristoylcyclopentenone,dimethoxyacetylcyclopentenone, dimethylfumarylcyclopentenone,dimethylmaleylcyclopentenone, di-2-hexenoylcyclopentenone,di-3-octenoylcyclopentenone and dibenzoylcyclopentenone.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof used in the present invention has an immunoregulatory activity,for example, an activity of inhibiting lymphocyte blastogenesis, anactivity of inhibiting mixed lymphocyte reaction, an activity ofactivating natural killer cells, an activity of activating cancercell-specific lymphocytes, an anti-inflammatory activity, for example,an activity of inhibiting carrageenan induced edema, an activity ofinhibiting delayed hypersensitivity, an activity of inhibiting tumornecrosis factor production, an activity of inhibiting topoisomerase, anactivity of inducing heat shock protein, an activity of inhibiting celladhesion, an antifungal activity or the like. Based on these activities,the compound is useful as a pharmaceutical composition for treating orpreventing a disease that requires immunoregulation for its treatment orprevention, a disease that requires suppression of inflammation for itstreatment or prevention, a disease that requires inhibition of tumornecrosis factor production for its treatment or prevention, a diseasethat requires inhibition of topoisomerase, a disease that requiresinduction of heat shock protein for its treatment or prevention, adisease that requires growth inhibition of fungus for its treatment orprevention, a disease that requires inhibition of cell adhesion for itstreatment or prevention or the like. Thus, an immunoregulatorycomposition, an anti-inflammatory composition, a composition forinhibiting tumor necrosis factor production, a composition forinhibiting topoisomerase, a composition for inducing heat shock protein,an antifungal composition and a composition for inhibiting cell adhesioncan be produced using at least one compound selected from the groupconsisting of a cyclopentenone derivative or an optical isomer thereof,and a salt thereof as an active ingredient.

Tumor necrosis factor was discovered as a factor that induceshemorrhagic necrosis at tumor sites. Tumor necrosis factor is currentlyrecognized as a cytokine that is involved widely in biological defenseand immunological mechanism on the basis of inflammation. Failure in theregulatory mechanism of tumor necrosis factor production brings varioustroubles to the host. Overproduction or unregulated production-of tumornecrosis factor is involved in a number of diseases. Such diseasesinclude rheumatoid arthritis, rheumatic myelitis, osteoarthritis, goutyarthritis, sepsis, septic shock, endotoxin shock, Gram-negativebacterial sepsis, toxic shock syndrome, cerebral malaria, chronicpneumonia, graft versus host reaction, allograft rejection, pyrexia andmyalgia due to an infectious disease such influenza, cachexia secondaryto infection or malignant tumor, cachexia secondary to human acquiredimmunodeficiency syndrome (AIDS), AIDS, AIDS-related syndrome, keloidformation, ulcerative colitis, multiple sclerosis, and autoimmunediseases such as autoimmune diabetes and systemic lupus erythematosus[Molecular Medicine, 33: 1010-1020, 1182-1189 (1996)]. The compositionfor inhibiting tumor necrosis factor production of the present inventionis useful for treating and preventing disease states mediated orworsened by tumor necrosis factor, for example, insulin-dependentdiabetes mellitus caused by tumor necrosis factor [Nature, 389: 610-614(1997)].

The present invention provides a method for regulating tumor necrosisfactor production in which at least one compound selected from the groupconsisting of a cyclopentenone derivative or an optical isomer thereofand a salt thereof is used as an active ingredient. Furthermore, thepresent invention provides a food or a drink for ameliorating diseasestates of a disease mediated or worsened by tumor necrosis factor or afood or drink for preventing such disease containing at least onecompound selected from the group consisting of a cyclopentenonederivative or an optical isomer thereof and a salt thereof.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has immunoregulatory activities such as an activity ofinhibiting lymphocyte blastogenesis and an activity of inhibiting mixedlymphocyte reaction. An immunoregulatory composition containing at leastone compound selected from these compounds as an active ingredient isuseful as a pharmaceutical composition for treating or preventing adisease due to abnormality in immune system or an immunological factoror a disease that requires immunopotentiation for its treatment orprevention.

Lymphocyte blastogenesis is a reaction in which mitogen binds to areceptor on the surface of a lymphocyte to activate the lymphocyte andpromotes its division and proliferation. Mixed lymphocyte reaction is areaction in which lymphocytes obtained from allogeneic animals are mixedand cultured, thereby inducing activation of lymphocytes due toincompatibility of major histocompatibility antigens to promote thedivision and proliferation of lymphocytes. The immunoregulatorycomposition inhibits these reactions and is particularly useful fortreating and preventing autoimmune diseases caused by abnormal increasein lymphocytes, for example, chronic diseases such as chronic nephritis,chronic colitis, type I diabetes and rheumatoid arthritis and is alsouseful for suppression of graft rejection.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has an activity of inhibiting carrageenan edema in foot. Thus,an anti-inflammatory composition containing at least one compoundselected from these compounds as an active ingredient is useful as apharmaceutical composition for treating or preventing a disease thatrequires suppression of inflammation for its treatment or prevention.

Carrageenan induced pedal edema model is a reaction in whichinflammatory cells such as macrophages and neutrophils are induced bysubcutaneously injecting an inflammatory agent carrageenan into a soleof foot, and inflammatory factors produced from these cells increasevascular permeability, resulting in edema. The activity of inhibitingedema of the anti-inflammatory composition is useful for treating orpreventing a disease that requires suppression of increase in vascularpermeability for its treatment or prevention, e.g., rheumatoidarthritis.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has an activity of inhibiting delayed hypersensitivity. Thus, ananti-inflammatory composition containing at least one compound selectedfrom these compounds as an active ingredient is useful as apharmaceutical composition for treating or preventing a disease thatrequires inhibition of delayed hypersensitivity, which is caused by aninfectious disease or the like, for its treatment or prevention.

Delayed hypersensitivity is an inflammatory reaction dependent oncellular immunity mediated by activated lymphocytes, monocytes,macrophages and the like. Cytokines produced from these cellsinfiltrating at inflammation sites increase vascular permeability,resulting in edema, granuloma, fibrosis and necrosis to cause severedisorders. Allergic dermatitis caused by delayed hypersensitivityaccounts for majority of contact dermatitis. In addition, delayedhypersensitivity causes allergy in which a bacterium, a virus or a drugacts as an antigen. A mouse model using sheep erythrocyte as an antigenis generally used for testing delayed hypersensitivity. A compound thatis effective in this model is considered to be useful for treating orpreventing the above-mentioned allergic diseases.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has an inhibitory activity on topoisomerase II. Topoisomerase IIis transiently expressed only during division phase in normal cells. Onthe other hand, it is highly expressed throughout the cell cycle whencells cancerate. Thus, a composition for inhibiting topoisomerasecontaining at least one compound selected from these compounds as anactive ingredient can be used as a carcinostatic. Furthermore, a methodfor inhibiting topoisomerase using at least one compound selected fromthese compounds as an active ingredient is useful for biochemicalstudies, screening of carcinostatics and the like.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has an activity of inhibiting cell adhesion. Thus, there isprovided a composition for inhibiting cell adhesion containing at leastone compound selected from these compounds as an active ingredient. Thecomposition for inhibiting cell adhesion of the present invention isuseful as a pharmaceutical composition for treating or preventing adisease that requires inhibition of cell adhesion for its treatment orprevention.

For example, metastasis of cancer is established by release of cancercells grown at primary lesion into blood vessels, migration tometastasis sites and infiltration into tissues. Among these, adhesion ofthe cancer cells to vascular endothelial cells is required for themigration of the cancer cells from the inside of blood vessels to themetastasis sites. ICAM-1, VCAM-1 and ELAM-1 on the vascular endothelialcells are known as adhesion molecules involved in metastasis of cancer.The corresponding ligands on the cancer cells have been identified asLFA-1, VLA-4 and sialyl Lewis X, respectively. These adhesion moleculesare often expressed on leukemia cells and considered to be involved inextravascular infiltration of leukemia cells. Thus, the composition forinhibiting cell adhesion of the present invention is expected to inhibitthe adhesion of cancer cells mediated by these adhesion molecules toinhibit metastasis of cancer.

Either of cyclopentenone and a compound produced from cyclopentenone andan SH group-containing compound as described in PCT/JP98/00815 also hasan activity of inhibiting cell adhesion. Thus, there is provided acomposition for inhibiting cell adhesion containing at least onecompound selected from these compounds as an active ingredient.

The composition for inhibiting cell adhesion provided by the presentinvention can be used for a method for inhibiting cell adhesion. Thismethod is useful for biochemical studies concerning cell adhesion andscreening of cell adhesion inhibitors or agents having an activity ofadhering cells.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has an activity of activating natural killer (NK) cells. Thus,there is provided a composition for activating NK cells containing atleast one compound selected from these compounds as an activeingredient. The composition for activating NK cells of the presentinvention is useful as a pharmaceutical composition for treating orpreventing a disease that requires activation of NK cells for itstreatment or prevention.

NK cells recognize cells infected with bacteria or viruses and cancercells, and eliminate these cells by cell membrane attack. Activation ofNK cells enhances the immunological protection mechanism in a livingbody. Thus, the composition for activating NK cells of the presentinvention is useful for treating or preventing a bacterial or viraldisease and cancer.

Either of cyclopentenone and a compound produced from cyclopentenone andan SH group-containing compound as described in PCT/JP98/00815 also hasan activity of activating NK cells. Thus, there is provided acomposition for activating NK cells containing at least one compoundselected from these compounds as an active ingredient.

The composition for activating NK cells provided by the presentinvention can be used for a method for activating NK cells. This methodis useful for biochemical studies concerning immunological protectionmechanism and screening of immunoprotective agents.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has an antifungal activity. Thus, an antifungal composition canbe produced by using at least one compound selected from these compoundsas an active ingredient and formulating it with a known pharmaceuticalcarrier.

A number of agents have been conventionally used for treating fungalinfection, including amphotericin B, flucytosine, miconazole andfluconazole. However, they have problems concerning the efficacy andtoxicity, or strains resistant thereto. In particular, less toxic agentseffective for systemic infection, which tends to increase recently, arefew. The antifungal component of the present invention is useful as anew type of an antifungal agent since it is less toxic.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has an activity of inducing heat shock protein. Thus, acomposition for inducing heat shock protein can be produced by using atleast one compound selected from these compounds as an activeingredient. The composition can be administered through a suitable routefor a disease that requires induction of heat shock protein for itstreatment or prevention.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has an activity of inducing heat shock proteins such as 70-kDaheat shock protein (HSP70). They have antiviral activities against RNAviruses and DNA viruses such as hepatitis virus, AIDS virus, influenzavirus, vesicular stomatitis virus and herpesvirus. Heat shock proteinsare involved in tumor immunity. Thus, these compounds are also effectivefor tumor immunity. In addition, these compounds also have activities ofbiological defense such as an anti-inflammatory activity. Thus, viraldiseases such as a cold due to influenza virus can be prevented ortreated by administering the compound of the present invention or anoptical isomer thereof, or a salt thereof.

Heat shock protein is a generic name of proteins of which the synthesisis induced when a cell or an individual is subjected to rapid change intemperature to one higher by 5 to 10° C. than normal temperature. Heatshock proteins are distributed in wide variety of organisms includingprokaryotes and higher eukaryotes. HSP90, HSP70, ubiquitin, HP26 and thelike are known as eukaryotic heat shock proteins. Among these, HSP70 isone of molecular chaperones which bind to proteins that have notcompletely folded or incompletely folded proteins and assist theirthree-dimensional structure formation. Amino acid sequences ofheat-shock proteins are well conserved in the course of evolution. HSP70is homologous to Escherichia coli DnaK protein. About ten HSP70 genesare present in human. Some of them are constitutively expressed whereasothers are induced by various stimuli. Synthesis of heat shock proteinis induced by various chemical substances and cell damages such asoxidative stress in addition to heat shock.

C. Amici et al. [Journal of Virology, 68:6890-6899 (1994)] reports thatcultivation of animal cells infected with Sendai virus in the presenceof prostaglandin A₁ having α,β-unsaturated carbonyl induces thesynthesis of SP70 and HSP90 and that synthesis of viral proteins issuppressed while the synthesis of HSP70 is induced. A. Rossi et al. [TheJournal of Biological Chemistry, 271:32192-32196 (1996)] reports that2-cyclopenten-1-one, like prostaglandin A₁, induces the synthesis ofHSP70 and suppresses the synthesis of proteins from vesicular stomatitisvirus.

For example, the ability of diisobutyrylcyclopentenone, a cyclopentenonederivative, to induce HSP70 is observed at a concentration of 1.25 μM.It is maximized at a concentration of 2.5 μM. This inducing ability isvery high as compared with that of 2-cyclopenten-1-one, which isrequired to be used at a concentration of several hundred μM forinducing HSP70.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof has antiviral activities against DNA viruses, RNA viruses,retroviruses and viroids base on the high activity of inducing heatshock protein. The viruses and viroids are exemplified by theabove-mentioned ones.

Generally, the immunoregulatory composition can be produced by using acompound selected from the group consisting of a cyclopentenonederivative or an optical isomer thereof, and a salt thereof as itsactive ingredient, and mixing it with a pharmaceutically acceptableliquid or solid carrier and, optionally, solvent, dispersing agent,emulsifier, buffering agent, stabilizer, excipient, binder,disintegrant, lubricant and the like to formulate it. The formulationmay be in a form of a solid preparation such as tablet, granule, powder,epipastic and capsule, or a liquid preparation such as normal solution,suspension and emulsion. In addition, the composition may be formulatedinto a dried preparation, which can be reconstituted as a liquidpreparation by adding an appropriate carrier before use.

The pharmaceutical carrier can be selected according to theabove-mentioned particular administration route and dosage form. For anoral preparation, starch, lactose, sucrose, mannit,carboxymethylcellulose, cornstarch, inorganic salts and the like areutilized, for example. Binder, disintegrant, surfactant, lubricant,fluidity-promoting agent, tasting agent, coloring agent, flavoring agentand the like can also be included in oral preparations.

A parenteral preparation can be prepared according to conventionalmethods by dissolving or suspending the active ingredient of the presentinvention, i.e., a compound selected from the group consisting of acyclopentenone derivative or an optical isomer thereof, and a saltthereof, in a diluent. The diluents include injectable distilled water,physiological saline, aqueous glucose solution, injectable vegetableoil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol andpolyethylene glycol. Optionally, sterilizer, stabilizer, osmoticregulator, smoothing agent and the like may be added to the solution orsuspension.

The immunoregulatory composition of the present invention isadministered through a suitable route for the dosage form of thecomposition. The administration route is not limited to a specific one.The composition can be administered internally or externally (ortopically) or by injection. The injectable preparation can beadministrated intravenously, intramuscularly, subcutaneously,intradermally and the like, for example. External preparations include asuppository.

A dosage of the immunoregulatory composition is appropriately determinedand varies depending on the particular dosage form, administration routeand purpose as well as age, weight and conditions of a patient to betreated. In general, a daily dosage for an adult person is 0.1 μg to 200mg/kg in terms of the amount of a compound selected from the groupconsisting of cyclopentenone or an optical isomer thereof, and a saltthereof contained in the formulation. Of course, the dosage can varydepending on various factors. Therefore, in some cases, a less dosagethan the above may be sufficient but, in other cases, a dosage more thanthe above may be required. The pharmaceutical composition of the presentinvention can be administrated orally as it is, or it can be taken dailyby adding to selected foods and drinks.

The composition for inhibiting tumor necrosis factor production, theanti-inflammatory composition, the composition for inhibitingtopoisomerase, the composition for inhibiting cell adhesion, thecomposition for inducing heat shock protein and the antifungalcomposition of the present invention can be formulated according to thesame manner as that described above with respect to the immunoregulatorycomposition. They can be administered through a suitable route for thedisease of interest. Of course, the dosage of the composition can varydepending on various factors. Therefore, in some cases, a less dosagethan the above may be sufficient but, in other cases, a dosage more thanthe above may be required. The pharmaceutical composition of the presentinvention can be administrated orally as it is, or it can be taken dailyby adding to selected foods and drinks.

The cyclopentenone derivative or an optical isomer thereof, or a saltthereof used in the present invention can be efficiently produced fromcyclopentenone and any one of carboxylic acids or reactive derivativesthereof.

There are provided diisobutyrylcyclopentenone (a product of reactionbetween cyclopentenone and isobutyric anhydride),dimethoxyacetylcyclopentenone (a product of reaction betweencyclopentenone and methoxyacetic acid), dimethylfumarylcyclopentenoneand dimethylmaleylcyclopentenone (products of reaction betweencyclopentenone and methylmaleic acid) according to the above-mentionedproduction method.

These compounds have a carcinostatic activity, an activity of inhibitingtopoisomerase and the like. Pharmaceutical compositions such as acarcinostatic composition can be produced by using these compounds oroptical isomers thereof, or salts thereof as their active ingredients.

The process for producing a food or a drink containing thecyclopentenone derivative or an optical isomer thereof, or a saltthereof obtained according to the present invention as its activeingredient is not limited to a specific one. Any processes includingcooking, processing and other generally employed processes for producinga food or a drink can be used as long as the resultant food or drinkcontains an effective amount of a compound having a physiologicalactivity selected from the group consisting of a cyclopentenonederivative or an optical isomer thereof, and a salt thereof. Afunctional food or drink such as an immunoregulatory food or drink canbe thus produced.

No toxicity is observed when a physiologically effective amount of thecyclopentenone derivative or an optical isomer thereof, or a saltthereof obtained according to the present invention is administered. Forexample, no death was observed when either one ofdipropionylcyclopentenone, dihexanoylcyclopentenone,di-2-hexenoylcyclopentenone, diisobutyrylcyclopentenone,dibenzoylcyclopentenone or optical isomers thereof, or salts thereof wasorally administered to a mouse at a single dosage of 100 mg/kg.

As described above, the cyclopentenone derivative or an optical isomerthereof, or a salt thereof can be conveniently produced and are veryuseful in a wide variety of fields including medicine and foods based ontheir various physiological functions.

The following examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof. Percent(%) in Examples means percent by weight.

EXAMPLE 1

(1) 10 g of D-glucuronic acid (Sigma, G 5269) was dissolved in 1 literof water. The solution was heated at 121° C. for 4 hours and thenconcentrated to a volume of about 10 ml under reduced pressure. 40 ml ofan upper layer of a mixture of butyl acetate:acetic acid:water=3:2:2 wasadded thereto and mixed. A supernatant obtained by centrifuging themixture was concentrated under reduced pressure to a volume of about 10ml.

The extract was applied to silica gel BW-300SP for column chromatography(2×28 cm, Fuji Sylysia) Separation was carried out using an upper layerof butyl acetate:acetic acid:water=3:2:2 as an eluent, at a pressure of0.2 kg/cm₂ using a compressor and at a flow rate of 5 ml/min. Eachfraction contained 10 ml of the fractionated eluate. A portion of eachfraction was analyzed on thin-layer chromatography. As a result, 61st to80th fractions contained cyclopentenone with high purity. Thesefractions were collected and concentrated under reduced pressure. Theconcentrate was extracted with 40 ml of chloroform. The extract wasconcentrated under reduced pressure to obtain 100 mg of cyclopentenone.

The preparation was separated on normal phase HPLC using Palpack Type Scolumn and detected on the basis of ultraviolet absorbance at 215 nm.This procedure confirmed that the preparation had a purity of 98%. 113.9mg of cyclopentenone obtained according to the method as described abovewas dissolved in 2.85 ml of ethanol. 3.85 ml of hexane/ethanol (94/6)was further added to the solution in ethanol to prepare 17 mg/mlcyclopentenone solution. This solution was filtrated through a 0.5-μmfilter to obtain a sample solution for optical resolution HPLC.

The sample solution was subjected to optical resolution HPLC under theconditions as described below. Fractions containing a (−) isomer and a(+) isomer of cyclopentenone were separately collected from the formerpeak and the latter peak, respectively. The fractions were evaporated todryness under reduced pressure to obtain 43.2 mg of the (−) isomer and43.0 mg of and the (+) isomer of cyclopentenone.

Conditions for optical resolution HPLC

Column: Chiralpack AS (Dicel Chemical Industries) 2.0 cm×25.0 cm;

Column temperature: 40° C.;

Mobile phase: hexan/ethanol (94/6);

Flow rate: 14.0 ml/min.;

Detection: UV 210 nm;

Amount of sample injected: 150 μl (2.55 mg).

Since both of the resulting (−) isomer and (+) isomer of cyclopentenonecontained an enantiomer at a concentration of about 1%, they wereoptically resolved again under the above-mentioned conditions. As aresult, 19.7 mg of the (−) isomer of cyclopentenone free of anenantiomer was obtained from 30.0 mg of the (−) isomer from the formerpeak. 27.7 mg of the (+) isomer of cyclopentenone free of an enantiomerwas obtained from 37.4 mg of the (+) isomer from the latter peak. Theelution time in the optical resolution HPLC for the (−) isomer ofcyclopentenone was 33 min. and that for the (+) isomer was 40 min.

(2) 1 ml of anhydrous pyridine (Nacalai Tesque, 295-26) and 0.1 ml ofacetic anhydride (Nacalai Tesque, 002-26) were added to 29.6 mg ofcyclopentenone obtained as described in Example 1-(1). The mixture wasstirred at is room temperature for 3 hours. The reaction mixture wasextracted with chloroform to obtain 36 mg of diacetylcyclopentenone.

Mass spectrometric analysis of the resulting diacetylcyclopentenone wascarried out using DX302 mass spectrometer (Nippon Denshi). Additionally,diacetylcyclopentenone was dissolved in CDCl₃ and subjected tostructural analysis by NMR using a nuclear magnetic resonance apparatusJNM-A500 (Nippon Denshi). The results are shown below. The chemicalshift values in ¹H-NMR are expressed assuming the chemical shift valueof chloroform as 7.24 ppm.

MS m/z 199 (M+H)⁺; ¹H-NMR; δ 2.12 (3H, S, —OCOCH₃), 2.16 (3H, S,—OCOCH₃), 5.16 (1H, d, J=3.0 Hz, H-5), 5.89 (1H, m, H-4), 6.40 (1H, d-d,J=1.5, 6.5 Hz, H-2), 7.43 (1H, d-d, J=2.5, 6.5 Hz, H-3).

(3) 15.9 mg of the (−) isomer of cyclopentenone obtained as described inExample 1-(1) was used to carry out the reaction as described in Example1-(2) to obtain 15.1 mg of a diacetyl (−) isomer of cyclopentenone.Similar results with those in Example 1-(2) were obtained when theisomer was subjected to structural analysis by mass spectrometry andnuclear magnetic resonance as described in Example 1-(2).

(4) 16.7 mg of the (+) isomer of cyclopentenone obtained as described inExample 1-(1) was used to carry out the reaction as described in Example1-(2) to obtain 18.8 mg of a diacetyl (+) isomer of cyclopentenone.Similar results with those in Example 1-(2) were obtained when theisomer was subjected to structural analysis by mass spectrometry andnuclear magnetic resonance as described in Example 1-(2).

(5) 44.3 mg of benzoic acid (Nacalai Tesque, 041-20), 7.5 mg ofdimethylaminopyridine (DMAP; Tokyo Kasei Kogyo, D1450), 51.0 mg ofN,N′-dicyclohexylcarbodiimide (DCC: Peptide Institute, 1001) and 5 ml ofchloroform were added to 13.8 mg of cyclopentenone. The mixture wasstirred on ice for 4 hours. A filtrate obtained by the filtration of thereaction mixture was applied to silica gel column (75 ml) and elutedwith chloroform to obtain fractions containing dibenzoylcyclopentenone.The solvent in the fractions was removed under reduced pressure, theresidue was dissolved in ethanol, and the solution was separated onsilica gel thin-layer chromatography using a 99:1 mixture of chloroformand methanol as a developing solvent. Silica gel at Rf=0.45-0.55 wasscraped from the thin layer and extracted with chloroform to obtain 3.2mg of dibenzoylcyclopentenone.

Structural analysis by mass spectrometry and nuclear magnetic resonanceof the thus obtained dibenzoylcyclopentenone was carried out asdescribed in Example 1-(2). The results are shown below.

MS m/z 323 (M+H)⁺; ¹H-NMR; δ 5.56 (1H, d, J=3.0 Hz, H-5), 6.30 (1H, m,H-4), 6.54 (1H, d-d, J=1.5, 6.5 Hz, H-2), 7.44 (4H, m, H of aromaticring), 7.58 (2H, m, H of aromatic ring), 7.64 (1H, d-d, J=2.0, 6.5 Hz,H-3), 8.06 (4H, m, H of aromatic ring).

(6) 22.1 mg of the (−) isomer of cyclopentenone, 71.9 mg of benzoicacid, 12.1 mg of DMAP and 80.3 mg of DCC were used to carry out thereaction as described in Example 1-(5) to obtain 19.2 mg of a dibenzoyl(−) isomer of cyclopentenone. Similar results with those in Example1-(5) were obtained when the isomer was subjected to structural analysisby mass spectrometry and nuclear magnetic resonance as described inExample 1-(5).

(7) 20.4 mg of the (+) isomer of cyclopentenone, 65.6 mg of benzoicacid, 11.0 mg of DMAP and 74.3 mg of DCC were used to carry out thereaction as described in Example 1-(5) to obtain 21.4 mg of a dibenzoyl(+) isomer of cyclopentenone. Similar results with those in Example1-(5) were obtained when the isomer was subjected to structural analysisby mass spectrometry and nuclear magnetic resonance as described inExample 1-(5).

(8) 30 mg of cyclopentenone, 10 mg of DMAP and 153 mg of hexanoic acid(Nacalai Tesque, 070-26) were dissolved in 5.9 ml of dichloromethane.108 mg of DCC was added thereto while cooling on ice. After reacting for1 hour, the reaction mixture was separated and purified on silica gelthin-layer chromatography using chloroform as a developing solvent.Silica gel at Rf=0.3-0.4 was scraped from the thin layer and extractedwith chloroform to obtain 11 mg of dihexanoylcyclopentenone.

The thus obtained dihexanoylcyclopentenone was dissolved in CDCl₃ forconfirmation by nuclear magnetic resonance (NMR) using a nuclearmagnetic resonance apparatus JNM-EX270 FT NMR system (Nippon Denshi).The chemical shift values in ¹H-NMR are expressed assuming the chemicalshift value of tetramethylsilane as 0 ppm.

The results are shown below.

¹H-NMR; δ 7.44 (1H, dd, J₂₋₃=6.27 Hz, J₃₋₄=1.98 Hz, H-3), 6.42 (1H, dd,J₂₋₃=6.27 Hz, J₃₋₄=1.32 Hz, H-2), 5.91 (1H, m, H-4), 5.16 (1H, d,J₄₋₅=2.97 Hz, H-5), 2.42 (2H, t, J=7.26 Hz), 2.38 (2H, t, J=7.76 Hz),1.65 (4H, m), 1.26 (8H, m), 0.88 (6H, t).

(9) 30 mg of cyclopentenone, 10 mg of DMAP and 301 mg of myristic acid(Tokyo Kasei Kogyo, M0476) were dissolved in 5.9 ml of dichloromethane.108 mg of DCC was added thereto while cooling on ice. After reacting for1 hour, the reaction mixture was separated on silica gel thin-layerchromatography using chloroform as a developing solvent. Silica gel atRf=0.45-0.55 was scraped from the thin layer and extracted withchloroform to obtain 53 mg of dimyristoylcyclopentenone.

Structural analysis by nuclear magnetic resonance of the thus obtaineddimyristoylcyclopentenone was carried out as described in Example 1-(8).The results are shown below.

¹H-NMR; δ 7.45 (1H, dd, J₂₋₃=5.94 Hz, J₃₋₄=2.31 Hz, H-3), 6.42 (1H, dd,J₂₋₃=5.31 Hz, J₃₋₄=1.32 Hz, H-2), 5.92 (1H, m, H-4), 5.16 (1H, d,J₄₋₅=2.64 Hz, H-5), 2.42 (2H, t, J=7.26 Hz), 2.38 (2H, t, J=7.91 Hz),1.63 (4H, m), 1.26 (32H, m), 0.88 (6H, t).

(10) 30 mg of cyclopentenone, 10 mg of DMAP and 190 mg of octanoic acid(Nacalai Tesque, 071-11) were dissolved in 5.9 ml of dichloromethane.108 mg of DCC was added thereto while cooling on ice. After reacting for1 hour, the reaction mixture was separated on silica gel thin-layerchromatography using chloroform as a developing solvent. Silica gel atRf=0.25-0.35 was scraped from the thin layer and extracted withchloroform to obtain 27 mg of dioctanoylcyclopentenone.

Structural analysis by nuclear magnetic resonance of the thus obtaineddioctanoylcyclopentenone was carried out as described in Example 1-(8).The results are shown below.

¹H-NMR; δ 7.44 (1H, dd, J₂₋₃=6.1 Hz, J₃₋₄=2.16 Hz, H-3), 6.41 (1H, dd,J₂₋₃=6.1 Hz, J₃₋₄=1.48 Hz, H-2), 5.92 (1H, m, H-4), 5.16 (1H, d,J₄₋₅=2.97 Hz, H-5), 2.42 (2H, t, J=7.59 Hz), 2.38 (2H, t, J=7.91 Hz),1.65 (4H, m), 1.29 (16H, m), 0.88 (6H, t).

(11) 30 mg of cyclopentenone, 10 mg of DMAP and 190 mg of 3-octenoicacid (Tokyo Kasei Kogyo, 00070) were dissolved in 5.9 ml ofdichloromethane. 108 mg of DCC was added thereto while cooling on ice.After reacting for 1 hour, the reaction mixture was separated on silicagel thin-layer chromatography using chloroform as a developing solvent.Silica gel at Rf=0.25-0.35 was scraped from the thin layer and extractedwith chloroform to obtain 25 mg of di-3-octenoylcyclopentenone.

Structural analysis by nuclear magnetic resonance of the thus obtaineddi-3-octenoylcyclopentenone was carried out as described in Example1-(8). The results are shown below.

¹H-NMR; δ 7.44 (1H, dd, J₂₋₃=6.27 Hz, J₃₋₄=2.32 Hz, H-3), 6.42 (1H, dd,J₂₋₃=6.27 Hz, J₃₋₄=1.49 Hz, H-2), 5.91 (1H, m, H-4), 5.55 (4H, m), 5.16(1H, d, J₄₋₅=2.97 Hz, H-5), 3.12 (4H, dd, J=12.85 Hz, J=6.59 Hz), 2.04(4H, m), 1.33 (8H, m), 0.89 (6H, t).

(12) 30 mg of cyclopentenone, 10 mg of DMAP and 115 mg of n-butyric acid(Tokyo Kasei Kogyo, B0754) were dissolved in 5.9 ml of dichloromethane.108 mg of DCC was added thereto while cooling on ice. After reacting for1 hour, the reaction mixture was separated on silica gel thin-layerchromatography using chloroform as a developing solvent. Silica gel atRf=0.20-0.30 was scraped from the thin layer and extracted withchloroform to obtain 16 mg of dibutyrylcyclopentenone.

Structural analysis by nuclear magnetic resonance of the thus obtaineddibutyrylcyclopentenone was carried out as described in Example 1-(8).The results are shown below.

¹H-NMR; δ 7.45 (1H, dd, J₂₋₃=6.27 Hz, J₃₋₄=2.13 Hz, H-3), 6.42 (1H, dd,J₂₋₃=6.27 Hz, J₃₋₄=1.65 Hz, H-2), 5.91 (1H, m, H-4), 5.16 (1H, d,J₄₋₅=2.64 Hz, H-5).

(13) 30 mg of cyclopentenone, 10 mg of DMAP and 226 mg of n-decanoicacid (Tokyo Kasei Kogyo, D0017) were dissolved in 5.9 ml ofdichloromethane. 108 mg of DCC was added thereto while cooling on ice.After reacting for 1 hour, the reaction mixture was separated on silicagel thin-layer chromatography using chloroform as a developing solvent.Silica gel at Rf=0.35-0.45 was scraped from the thin layer and extractedwith chloroform to obtain 35 mg of didecanoylcyclopentenone.

Structural analysis by nuclear magnetic resonance of the thus obtaineddidecanoylcyclopentenone was carried out as described in Example 1-(8).The results are shown below.

¹H-NMR; δ 7.44 (1H, dd, J₂₋₃=6.27 Hz, J₃₋₄=1.97 Hz, H-3), 6.42 (1H, dd,J₂₋₃=6.27 Hz, J₃₋₄=1.3 Hz, H-2), 5.91 (1H, m, H-4), 5.15 (1H, d,J₄₋₅=2.97 Hz, H-5), 2.42 (2H, t, J=7.24 Hz), 2.38 (2H, t, J=7.91 Hz),1.65 (4H, m), 1.26 (24H, m), 0.88 (6H, t).

(14) 30 mg of cyclopentenone, 16 mg of DMAP, 66 mg of triethylamine(Tokyo Kasei Kogyo, T0424) and 122 mg of n-valeric anhydride (TokyoKasei Kogyo, V0006) were dissolved in 5.9 ml of dichloromethane. Themixture was reacted on ice for 1 hour. The reaction mixture wasdeveloped on silica gel thin-layer chromatography usingchloroform:methanol 200:1 as a developing solvent. Silica gel atRf=0.7-0.8 was scraped from the thin layer and extracted with chloroformto obtain 39 mg of divalerylcyclopentenone.

Structural analysis by nuclear magnetic resonance of the thus obtaineddivalerylcyclopentenone was carried out as described in Example 1-(8).The results are shown below.

¹H-NMR; δ 7.45 (1H, dd, J2-3=6.11 Hz, J3-4=1.66 Hz, H-3), 6.42 (1H, dd,J2-3=6.11 Hz, J3-4=1.66 Hz, H-2), 5.91 (1H, m, H-4), 5.16 (1H, d,J4-5=2.97 Hz, H-5), 2.43 (2H, dd, J=7.59, 7.59 Hz), 2.39 (2H, dd,J=7.59, 7.59 Hz), 1.65 (4H, m), 1.38 (4H, m), 0.93 (6H, dd, J=7.26, 7.26Hz).

(15) 30 mg of cyclopentenone, 16 mg of DMAP, 66 mg of triethylamine and86 mg of propionic anhydride (Tokyo Kasei Kogyo, P0513) were dissolvedin 5.9 ml of dichloromethane. The mixture was reacted on ice for 1 hour.The reaction mixture was developed on silica gel thin-layerchromatography using chloroform:methanol=200:1 as a developing solvent.Silica gel at Rf=0.5-0.6 was scraped from the thin layer and extractedwith chloroform to obtain 31 mg of dipropionylcyclopentenone.

Structural analysis by nuclear magnetic resonance of the thus obtaineddipropionylcyclopentenone was carried out as described in Example 1-(8).The results are shown below.

¹H-NMR; δ 7.45 (1H, dd, J2-3=6.27 Hz, J3-4=2.15 Hz, H-3), 6.42 (1H, dd,J2-3=6.27 Hz, J3-4=1.49 Hz, H-2), 5.91 (1H, m, H-4), 5.16 (1H, d,J4-5=2.97 Hz, H-5), 2.46 (2H, dd, J=15.01, 7.59 Hz), 2.42 (2H, dd,J=15.01, 7.59 Hz), 1.18 (6H, dd, J=7.59, 7.59 Hz).

(16) 2 g of cyclopentenone, 733 mg of DMAP, 4.1 ml of trans-2-hexenoicacid (Tokyo Kasei Kogyo, H0383) and 5.57 g of DCC were dissolved in 200ml of dichloromethane. The mixture was reacted at room temperature for 2hours. The reaction mixture was subjected to silica gel columnchromatography using hexane:ethyl acetate=8:1 as a solvent to obtain afraction that results in a single spot on silica gel thin-layerchromatography. The fraction was concentrated under reduced pressure toobtain about 900 mg of oil of di-2-hexenoylcyclopentenone.

Structural analysis by nuclear magnetic resonance of the thus obtaineddi-2-hexenoylcyclopentenone was carried out as described in Example1-(8). The results are shown below.

¹H-NMR; δ 0.92 (6H, m, 11-H+11′-H), 1.48 (4H, m, 10-H+10′-H), 2.18 (4H,m, 9-H, 9′-H), 5.22 (1H, d, J=3.0 Hz, 5-H), 5.85 (2H, m, 7-H+7′-H), 5.98(1H, m, 4-H), 6.41 (1H, dd, J=1.0, 6.0 Hz, 2-H), 7.04 (2H, m, 8-H+8′-H),7.47 (1H, dd, J=2.0, 6.0 Hz, 3-H).

The positions of carbons in the 2-hexenoyl group attached at 5-positionof cyclopentenone were defined as 6-position to 11-position startingfrom the carbonyl group. The positions of carbons in the 2-hexenoylgroup attached at 4-position of cyclopentenone were defined as6′-position to 11′-position starting from the carbonyl group.

(17) 1.2 g of cyclopentenone was dissolved in 200 ml of dichloromethane.1.7 ml of isobutyric anhydride (Nacalai Tesque), 427 mg of DMAP and 1.46ml of triethylamine (Nacalai Tesque) were added thereto. The mixture wasreacted at room temperature for 1 hour and concentrated under reducedpressure. The residue was dissolved in ethyl acetate and washed with 10%citric acid and saturated aqueous sodium hydrogencarbonate solution. Thesolution was concentrated under reduced pressure. The concentrate wasseparated on silica gel column chromatography using hexane:ethylacetate=8:1 as a developing solvent to obtain a fraction that results ina spot at Rf=0.2 on silica gel thin-layer chromatography usinghexane:ethyl acetate=6:1 as a developing solvent. The solvent in thefraction was removed by evaporating under reduced pressure to obtain 470mg of oil containing diisobutyrylcyclopentenone with high purity.

Structural analysis by nuclear magnetic resonance of the thus obtaineddiisobutyrylcyclopentenone dissolved in heavy chloroform was carried outas described in Example 1-(2). The results are shown below.

¹H-NMR; δ 1.18 (12H, m, 7-H, 8-H, 10-H, 11-H), 2.61 (2H, m, 6-H, 9-H),5.10 (1H, d, J=3.0 Hz, 5-H), 5.88 (1H, m, 4-H), 6.39 (1H, dd, J=1.5, 6.0Hz, 2-H), 7.41 (1H, dd, J=2.5, 6.0 Hz, 3-H).

The results are expressed assuming the chemical shift value of theresidual proton of heavy chloroform as 7.24 ppm.

The ¹H-NMR spectrum of diisobutyrylcyclopentenone is illustrated in FIG.1. In FIG. 1, the horizontal axis represents the chemical shift value(ppm) and the vertical axis represents the signal intensity.

The numbers for signal identification in ¹H-NMR are as indicated informula [III] below.

(18) 1.5 g of cyclopentenone was dissolved in 200 ml of dichloromethane.2.7 g of methoxyacetic acid (Nacalai Tesque), 794 mg of DMAP and 5.36 gof dicyclohexylcarbodiimide (DCC; Nacalai Tesque) were added thereto.The mixture was reacted at room temperature for 2 hours and concentratedunder reduced pressure. The residue was dissolved in ethyl acetate andwashed with 10% citric acid and saturated aqueous sodiumhydrogencarbonate solution. The solution was concentrated under reducedpressure. The concentrate was separated on silica gel columnchromatography using hexane:ethyl acetate=2:3 as a developing solvent toobtain a fraction that results in a spot at Rf=0.34 on silica gelthin-layer chromatography using hexane:ethyl acetate=1:1 as a developingsolvent. The solvent in the fraction was removed by evaporating underreduced pressure to obtain 300 mg of oil containingdimethoxyacetylcyclopentenone with high purity.

Structural analysis by nuclear magnetic resonance of the thus obtaineddimethoxyacetylcyclopentenone dissolved in heavy chloroform was carriedout as described in Example 1-(2). The results are shown below.

¹H-NMR; δ 3.45 (6H, s, 7-H, 9-H), 4.13 (4H, m, 6-H, 8-H), 5.30 (1H, d,J=3.0 Hz, 5-H), 5.99 (1H, m, 4-H), 6.44 (1H, dd, J=1.5, 6.5 Hz, 2-H),7.46 (1H, dd, J=2.0, 6.5 Hz, 3-H).

The results are expressed assuming the chemical shift value of theresidual proton of heavy chloroform as 7.24 ppm.

The ¹H-NMR spectrum of dimethoxyacetylcyclopentenone is illustrated inFIG. 2. In FIG. 2, the horizontal axis represents the chemical shiftvalue (ppm) and the vertical axis represents the signal intensity.

The numbers for signal identification in ¹H-NMR are as indicated informula [IV] below.

(19) 1.1 g of cyclopentenone was dissolved in 200 ml of dichloromethane.3.4 g of methylmaleic acid, 610 mg of DMAP and 4.12 g ofdicyclohexylcarbodiimide were added thereto. The mixture was reacted atroom temperature for 2 hours and concentrated under reduced pressure.The residue was dissolved in ethyl acetate and washed with 10% citricacid and saturated aqueous sodium hydrogencarbonate solution. Thesolution was concentrated under reduced pressure. The concentrate wasseparated on silica gel column chromatography using hexane:ethylacetate=3:2 as a developing solvent to obtain a fraction that results ina spot at Rf=0.6 and a fraction that results in a spot at Rf=0.45 onsilica gel thin-layer chromatography using hexane:ethyl acetate=1:1 as adeveloping solvent.

The solvent in the fractions was removed by evaporating under reducedpressure to obtain 300 mg of solid containingdimethylfumarylcyclopentenone with high purity from the Rf=0.6 fractionand 300 mg of oil containing dimethylmaleylcyclopentenone with highpurity from the Rf=0.45 fraction.

Structural analysis by nuclear magnetic resonance of the productsdissolved in heavy chloroform was carried out as described in Example1-(2). The results are shown below.

¹H-NMR;

Dimethylfumarylcyclopentenone

δ 3.80 (6H, s, 10-H, 15-H), 5.31 (1H, d, J=3.0 Hz, 5-H), 6.03 (1H, m,4-H), 6.48 (1H, dd, J=1.0, 6.0 Hz, 2-H), 6.90 (4H, m, 7-H, 8-H, 12-H,13-H), 7.50 (1H, dd, J=2.0, 6.0 Hz, 3-H).

Dimethylmaleylcyclopentenone

δ 3.76 (6H, s, 10-H, 15-H), 5.31 (1H, d, J=3.0 Hz, 5-H), 6.07 (1H, m,4-H), 6.31 (4H, m, 7-H, 8-H, 12-H, 13-H), 6.44 (1H, dd, J=1.5, 6.0 Hz,2-H), 7.58 (1H, dd, J=2.0, 6.0 Hz, 3-H).

The results are expressed assuming the chemical shift value of theresidual proton of heavy chloroform as 7.24 ppm.

The ¹H-NMR spectrum of dimethylfumarylcyclopentenone is illustrated inFIG. 3. The ¹H-NMR spectrum of dimethylmaleylcyclopentenone isillustrated in FIG. 4. In FIGS. 3 and 4, the horizontal axes representthe chemical shift value (ppm) and the vertical axes represent thesignal intensity.

The numbers for signal identification in ¹H-NMR fordimethylfumarylcyclopentenone are as indicated in formula [V] below.

The numbers for signal identification in ¹H-NMR fordimethylmaleylcyclopentenone are as indicated in formula [IV] below.

(20) 100 μl of 1M aqueous cyclopentenone solution and 500 μl of 200 mMaqueous glutathione (reduced; Nacalai Tesque, 170-10) solution (pH 3.0)were mixed together. The mixture was reacted at 60° C. for 5 hours. Thereaction mixture was filtrated through a 0.5-μm Cosmonice filter andseparated on HPLC under the following conditions.

Column: TSKgel ODS-80Ts (5 μm) 20 mm×25 cm;

Mobile Phase A: 0.1% aqueous TFA solution;

B: aqueous solution containing 0.1% TFA/50% acetonitrile;

Flow rate: 7.5 ml/min.;

Gradient: Mobile Phase A (10 min.)→Mobile Phase A to A:B=1:1 (55min.)→A:B=1:1 to Mobile Phase B (15 min.);

Detection: absorbance at 220 nm.

200 μl of the reaction mixture was subjected to HPLC. Peaks at retentiontime of 35.7 min. and 36.1 min. were collected and evaporated to drynessunder reduced pressure to obtain 5.5 mg of dry solid.

The structure of the dry solid was analyzed. Measurements were carriedout using JNM-A500 (Nippon Denshi) for nuclear magnetic resonance (NMR)spectrum, DX302 mass spectrometer (Nippon Denshi) for mass spectrum(MS), UV-2500 spectrophotometer (Shimadzu) for ultraviolet (UV)absorption spectrum and FTIR-8000PC infrared spectrometer (Shimadzu) forinfrared absorption (IR) spectrum, respectively. The results are shownbelow.

¹H-NMR; δ 2.09 (2H, m, 5′-H), 2.28 (1H, dd, J=13.0, 20.0 Hz, 5-H), 2.44(2H, m, 4′-H), 2.78 (1H, dd, J=8.5, 14.0, 1′-H), 2.85 or 2.89 (1H, dd,J=3.0, 6.0 Hz, 5-H), 2.92 or 2.95 (1H, dd, J=1.0, 5.5 Hz, 1′-H), 3.86(2H, S, 9′-H), 3.95 (2H, m, 4-H, 6′-H), 4.46 (1H, m, 2′-H), 6.47 or 6.49(1H, d, J=3.0 Hz, 3-H)

The sample was dissolved in 0.1 N DCl solution in heavy water. Theresults are expressed assuming the chemical shift value of HOD as 4.65ppm.

¹³C-NMR; δ 26.3 (5′-C), 31.7 (4′-C), 31.9 or 32.1 (1′-C), 39.3 (4-C),41.9 (9′-C), 42.2 or 42.3 (5-C), 53.3 (6′-C), 54.1 (2′-C), 133.5 (3-C),154.4 (2-C), around 173 (3′-C, 7′-C, 8′-C, 10′-C), 205.8 (1-C).

The sample was dissolved in 0.1 N DCl solution in heavy water. Theresults are expressed assuming the chemical shift value of dioxane as67.4 ppm.

The numbers for peak identification in ¹H-NMR and ¹³C-NMR are asindicated in formula [VII] below.

FAB-MS; m/z 404 (M+H)⁺, 426 (M+Na)⁺.

Glycerol was used for matrix.

UV λ_(max) 251 nm (water); IR ν^(KBr) _(max) cm⁻¹ 2949, 1710, 1660,1539, 1404, 1203. Measurement was carried out according to diffusereflectance method.

These results revealed that the dry solid was2-hydroxy-4-glutathion-S-yl-2-cyclopenten-1-one (hereinafter simplyreferred to as GM).

EXAMPLE 2

HL-60 (ATCC CCL-240) cells were cultured at 37° C. in RPMI 1640 medium(Bio Whittaker) containing 10% fetal calf serum (FCS; JRH) which hadbeen treated at 56° C. for 30 minutes and suspended in RPMI 1640 mediumat a concentration of 2.5×10⁵ cells/5 ml.

10 μl each of solutions of diisobutyrylcyclopentenone,dimethoxyacetylcyclopentenone, dimethylfumarylcyclopentenone ordimethylmaleylcyclopentenone in 70% ethanol in water diluted to varyingconcentrations was added to 5 ml of the suspension. The mixtures wereincubated at 37° C. for 24 hours in the presence of 5% Co₂. 10 μl ofaqueous actinomycin D (Sigma) solution (0.5 mg/ml), which is known as areagent that induces apoptosis, was used in place of the sample and themixture was incubated under the same conditions for confirmation.

The cultured cells were examined under an optical microscope.Condensation of nuclei, shrinking of cells and formation of apoptoticbodies were observed for the cells cultured with the addition of thesamples and actinomycin D. No such phenomenon was observed for thecontrol cells cultured with the addition of 10 μl of 70% aqueous ethanolsolution.

Furthermore, a portion of the cells cultured as described above wasstained with 0.4% Trypan Blue and examined under an optical microscope.The number of viable cells which were not stained and the number of deadcells which were stained blue were counted. The concentration of each ofthe samples that results in a viability of 50% (Viability₅₀ μM) wasdetermined. The results are shown in Table 1.

TABLE 1 Sample Viability₅₀ (μM) Diisobutyrylcyclopentenone 8.8Dimethoxyacetylcyclopentenone 14 Dimethylfumarylcyclopentenone 2.9Dimethylmaleylcyclopentenone 3.4

As described above, each compound exhibited an antiproliferationactivity against tumor cells and an apoptosis-inducing activity. Inaddition, optical isomers of the respective compounds and salts thereofexhibited similar activities.

EXAMPLE 3

(1) 2 μl of topoisomerase II (TopoGEN; 2 units/μl), 2 μl of 10-foldconcentrated buffer [0.5 M Tris-HCl (pH 8.0), 1.2 M KCl, 0.1 M MgCl₂,2.5 mM adenosine triphosphate, 5 mM dithiothreitol], 2 μl of 0.1% bovineserum albumin (Takara Shuzo), 11 μl of distilled water and 2 μl ofdistilled water (control) or 2 μl of one of aqueous solutions ofdipropionylcyclopentenone, diisobutyrylcyclopentenone,dibenzoylcyclopentenone, dihexanoylcyclopentenone,di-2-hexenoylcyclopentenone, dimethoxyacetylcyclopentenone,dimethylfumarylcyclopentenone or dimethylmaleylcyclopentenone at varyingconcentrations were mixed together. 1 μl of 0.25 μg/μl pBR322 DNA(Takara Shuzo) was added thereto. The resulting mixture was reacted at37° C. After reacting for 30 minutes, 2 μl of an aqueous solutioncontaining 1% sodium dodecyl sulfate, 50% glycerol and 0.02% BromophenolBlue was added thereto to stop the reaction.

20 μl of the reaction mixture was applied to 1% agarose gel preparedusing agarose L03 (Takara Shuzo) and TAE buffer [40 mM Tris, 5 mM sodiumacetate, 1 mm ethylenediaminetetraacetic acid disodium salt (EDTA);adjusted to pH 7.8 using acetic acid] and electrophoresed in TAE buffer.After electrophoresis, the gel was soaked in a 1 μg/ml aqueous ethidiumbromide solution. The gel was exposed to ultraviolet rays to visualizethe electophoretic pattern of DNA. The form of DNA is completely changedfrom superhelical type to relaxed type for a control to which water isadded, whereas the change from superhelical type to relaxed type ispartially or completely inhibited if topoisomerase II activity isinhibited.

As a result, the form of DNA was completely changed from superhelicaltype to relaxed type for a control to which water was added. On theother hand, the change in the form of the DNA from superhelical type torelaxed type was partially or completely inhibited bydipropionylcyclopentenone at a concentration of 0.1 μM or more,diisobutyrylcyclopentenone at a concentration of 1 μM or more,dibenzoylcyclopentenone at a concentration of 1 μM or more,dihexanoylcyclopentenone at a concentration of 10 μM or more,di-2-hexenoylcyclopentenone at a concentration of 10 μM or more,dimethoxyacetylcyclopentenone at a concentration of 50 μM or more,dimethylfumarylcyclopentenone at a concentration of 10 μM or more ordimethylmaleylcyclopentenone at a concentration of 5 μM or more,confirming the activity of inhibiting topoisomerase II of each of thecyclopentenone derivatives.

(2) The activity of inhibiting topoisomerase I of each of thecyclopentenone derivatives was determined as described in Example 3-(1)except that topoisomerase I (TopoGEN; 0.01 unit/μl) was used in place oftopoisomerase II and a solution containing 100 mM Tris-HCl (pH 7.9), 10mM EDTA, 1 mM spermidine and 50% glycerol was used as 10-foldconcentrated buffer.

As a result, the form of DNA was completely changed from superhelicaltype to relaxed type for a control to which water was added. On theother hand, the change in the form of the DNA from superhelical type torelaxed type was partially or completely inhibited bydipropionylcyclopentenone at a concentration of 1000 μM or more,diisobutyrylcyclopentenone at a concentration of 500 μM or more,dibenzoylcyclopentenone at a concentration of 50 μM or more,dihexanoylcyclopentenone at a concentration of 1000 μM or more,di-2-hexenoylcyclopentenone at a concentration of 500 μM or more,dimethoxyacetylcyclopentenone at a concentration of 1000 μM or more,dimethylfumarylcyclopentenone at a concentration of 1000 μM or more ordimethylmaleylcyclopentenone at a concentration of 1000 μM or more,confirming the activity of inhibiting topoisomerase I of each of thecyclopentenone derivatives.

As described above, each of the above-mentioned cyclopentenonederivatives as well as other cyclopentenone derivatives as described inExample 1 exhibited an inhibitory activity on topoisomerase II.Topoisomerase II is transiently expressed only during division phase innormal cells, whereas it is highly expressed throughout the cell cyclewhen cells cancerate. The inhibitory activity on topoisomerase II wasstronger than that on topoisomerase I of which the expression level andactivity are increased upon canceration.

In addition, optical isomers of the respective compounds and saltsthereof exhibited similar inhibitory activities specific fortopoisomerase II.

EXAMPLE 4

(1) 5 ml of RPMI 1640 medium containing 10% fetal calf serum and HL-60(ATCC CCL-240) cells at a concentration of 2×10⁵ cells/ml was placed ineach well of a 6-well plate. The plate was incubated at 37° C. for 24hours in the presence of 5% CO₂. Dipropionylcyclopentenone,diisobutyrylcyclopentenone or di-2-hexenoylcyclopentenone at a finalconcentration of 0, 0.63, 1.3, 2.5, 5, 10 or 20 μM was then addedthereto. The incubation was continued for additional 6 hours.

After incubation, the cell number was counted. The cells were harvestedby centrifugation, washed with PBS to prepare cells treated with one ofthe samples. Cells that were cultured in the same manner after heated at45° C. for 10 minutes were also prepared.

These treated cells were used for SDS-PAGE according to the method asdescribed in Molecular Cloning [Cold Spring Harbor Laboratory Press(1989)]. The treated cells were suspended in SDS-PAGE Sample buffer at aconcentration of 2.5×10⁶ cells/ml. The cell suspensions were treated at100° C. for 10 minutes. 5 μl each of the cell suspensions was applied totwo SDS-PAGE gels (5% stacking gel, 10% separation gel) andelectrophoresed. One of the gels was subjected to Coomassie staining.The other gel was blotted onto a polyvinylidene difluoride transfermembrane (Immobilon™, Millipore, Cat. #IPVH000-10). The membrane wasblocked at 4° C. overnight using Block Ace (Dainippon Pharmaceutical,Cat. #UK-B25).

The blocked membrane was reacted with a monoclonal antibodyHSP72/73(Ab-1) (Oncogene Research Products, Cat. #HSP01), whichspecifically reacts with heat-inducible 70-kDa heat shock protein. Themembrane was washed with TBS containing 0.05% Tween 20 followed by TBS.The membrane was then reacted with a peroxidase-conjugated secondaryantibody HRP-Rabbit Anti-Mouse IgG (H+L) (Zymed Laboratories, Inc., Cat.#61-6520), and then washed as described above. The membrane reacted withthe primary and secondary antibodies was reacted with a chemiluminolreagent Renaissance™ (Dupont NEN, Cat. #NEL-100). The membrane was thenexposed to an X-ray film to confirm the induction of 70-kDa heat shockprotein.

As a result, the induction of 70-kDa heat shock protein was observed.The degree of the induction is shown in Table 2. In Table 2, +represents the induction level. Increased number of + means increasedinduction. − means that no induction was observed. ± means that a slightinduction was observed.

TABLE 2 Cyclopentenone Concentration (μM) derivative 0.63 1.3 2.5 5 1020 Dipropionyl- − ± + +++ ++ ++ cyclopentenone Diisobutyryl- ± + ++++++ + + cyclopentenone Di-2-hexenoyl- ± + ++ +++ ± − cyclopentenone

The result for untreated control was −, and the result for the cellscultured after heating at 45° C. for 10 minutes was ++. As describedabove, each of the above-mentioned compounds exhibited an activity ofinducing heat shock protein at a low concentration. In addition, opticalisomers of the respective compounds and salts thereof exhibited similaractivities. Furthermore, other cyclopentenone derivatives or opticalisomers thereof, or salts thereof exhibited similar activities.

EXAMPLE 5

Dipropionylcyclopentenone, diisobutyrylcyclopentenone anddi-2-hexenoylcyclopentenone were used to examine their antifungalactivities against Candida albicans TIMM0136.

Candida albicans cells were cultured overnight in YNBG medium containing0.67% Yeast Nitrogen Base (Difco) and 1.0% glucose (seed culture). Theculture was then diluted with fresh Yeast Nitrogen Base medium to aconcentration of 1×10⁶ cells/ml. 100 μl of the dilution was dispensedinto each well of a 96-well microtiter plate.

10 μl each of solutions of dipropionylcyclopentenone,diisobutyrylcyclopentenone or di-2-hexenoylcyclopentenone in 70% ethanolat a concentration of 100 μg/ml or 500 μg/ml, or 10 μl of 70% ethanolsolution was added to the well. The plate was incubated without shakingat 30° C. for 48 hours (main culture).

Dipropionylcyclopentenone, diisobutyrylcyclopentenone ordi-2-hexenoylcyclopentenone suppressed the growth of Candida albicans ata concentration of 500 μg/ml. The minimum growth inhibitoryconcentration of each compound was 500 μg/ml. Thus, these compounds areuseful as active ingredients for antifungal compositions. In addition,optical isomers of the cyclopentenone derivatives and salts thereofexhibited similar activities.

EXAMPLE 6

Human vascular endothelial cells (sold by Sanko Junyaku) suspended inRPMI-1640 medium (Gibco) containing 10% FCS (HyClone) at a concentrationof 1×10⁵ cells/ml. 100 μl/well of the suspension was seeded into a96-well microtiter plate.

100 U/ml of human recombinant TNF-α (Promega) was added to monolayer ofvascular endothelial cells after incubation at 37° C. for 2 days in a 5%CO₂ incubator. The plate was incubated 37° C. for 6 hours in a 5% CO₂incubator to prepare vascular endothelial cells stimulated with TNF-α.

On the other hand, cyclopentenone, GM or dipropionylcyclopentenone at avarying concentration was added to HL-60 cells suspended in RPMI-1640medium containing 10% FCS at a concentration of 1×10⁶ cells/ml. Thecells were cultured at 37° C. for 3 hours in a 5% CO₂ incubator. Aftercultivation, 5 μM of 5(-and-6)-carboxyfluorescein diacetate, succinimideester (Molecular Probe) as a fluorescent agent was added thereto andreacted at 37° C. for 10 minutes. The cells were washed twice withRPMI-1640 medium and suspended in RPMI-1640 medium containing 10% FCS toprepare a suspension of fluorescence-labeled HL-60 cells at aconcentration of 1×10⁶ cells/ml.

100 μl/well of the fluorescence-labeled HL-60 cells were overlaid ontothe vascular endothelial cells stimulated with TNF-α. Cyclopentenone, GMor dipropionylcyclopentenone at a varying concentration was addedthereto. The plate was incubated at 37° C. for 20 minutes in a 5% CO₂incubator for adhesion reaction between vascular endothelial cells andHL-60 cells.

After the adhesion reaction, the plate was washed to remove non-adhesivecells. 1% Triton X (Nacalai Tesque) was added thereto to destroyadhesive cells. The fluorescence intensity was measured using filtersfor 485/22 nm (excitation) and 530/25 nm (emission).

The adhesion rate which represents the ratio of cells adhered tovascular endothelial cells was determined defining the fluorescenceintensity for 1×10₅ fluorescence-labeled cells as 100%.

The results are shown in Table 3. The rate of adhesion of vascularendothelial cells to HL-60 was increased when they were stimulated withTNF-α. This increase in adhesion rate was suppressed by cyclopentenone,GM or dipropionylcyclopentenone at a concentration ranging from 10⁻⁷ to10⁻⁴ M in a dose-dependent manner. Thus, each of cyclopentenone, GM anddipropionylcyclopentenone exhibited an activity of inhibiting adhesionbetween cancer cells and vascular endothelial cells, which is requiredfor the inhibition of metastasis of cancer. In addition, optical isomersof the respective compounds and salts thereof exhibited similaractivities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

TABLE 3 Concentration Adhesion rate Test substance (μM) (%) ControlUnstimulated 3.6 Stimulated with TNF-α 50.9 Cyclopentenone 0.1 51.7 146.0 10 22.2 100 17.9 GM 0.1 53.9 1 48.3 10 23.9 100 16.3Dipropionylcyclopentenone 0.1 49.2 1 35.1 10 15.3 100 15.5

EXAMPLE 7

ddY mice (female, 7 weeks old) were purchased from Japan SLC andpre-bred 1 week before using in experiments.

1×10⁶ Ehrlich ascites carcinoma cells were intraperitoneally inoculatedto each mouse. 10 mg/kg of cyclopentene, 30 mg/kg of GM or 10 mg/kg ofdipropionylcyclopentene was intraperitoneally administered to the mouseon the day after the inoculation. Saline was administered to a controlmouse.

A spleen was taken out from the mouse 10 days after the inoculation withcancer cells, finely minced and suspended in RPMI-1640 medium (Gibco)containing 10% FCS (HyClone) to obtain a single cell suspension.Adhesive cells in the cell suspension adhered to a plastic Petri dishwere removed and non-adhesive cells were used as spleen lymphocytes.

The spleen lymphocytes were suspended in RPMI-1640 medium containing 10%FCS at a concentration of 2×10⁶/ml. 100 μl/well of the suspension wasseeded into a microtiter plate.

Stimulated cells were prepared as follows. Mitomycin C (Kyowa HakkoKogyo) was added to a concentration of 50 μg/ml to Ehrlich ascitescarcinoma cells suspended in RPMI-1640 medium at a concentration of2×10⁶ cells/ml. The cells were treated at 37° C. for 30 minutes, washedtwice and then suspended in RPMI-1640 medium containing 10% FCS toprepare stimulated cells at a concentration of 2×10⁶ cells/ml. 100 μl ofthe thus prepared stimulated cells was overlaid onto each well of theplate into which 100 μl/well of the spleen lymphocytes had been added.The plate was incubated at 37° C. for 5 days in a 5% CO₂ incubator. 37kBq of ³H-thymidine (Daiichi Pure Chemicals) was added to the well topulse-label the cells on the day before the completion of thecultivation. After cultivation, the cells were harvested on a glassfilter to measure the radioactivity.

The results are shown in Table 4. The growth of spleen lymphocytesobtained from mice administered with cyclopentenone, GM ordipropionylcyclopentenone were significantly enhanced by stimulationwith cancer cells, suggesting that lymphocyte specifically reactive withcancer cells were induced. In addition, optical isomers of therespective compounds and salts thereof exhibited similar activities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

TABLE 4 Stimulation ³H-thymidine Dose with uptake Test substance (mg/kg)cancer cell (CPM) Control − 10492 + 8806 Cyclopentenone 10 − 9680 +25756 GM 30 − 8700 + 36291 Dipropionyl- 10 − 9250 cyclopentenone + 20748

EXAMPLE 8

ddY mice (female, 7 weeks old) were purchased from Japan SLC andpre-bred for 1 week before using in experiments.

1×10⁶ Ehrlich ascites carcinoma cells were intraperitoneally inoculatedto each mouse. 10 mg/kg of cyclopentenone, 30 mg/kg of GM or 10 mg/kg ofdipropionylcyclopentenone was intraperitoneally administered to themouse on the day after the inoculation. Saline was administered to acontrol mouse.

A spleen was taken out from the mouse 10 days after the inoculation withcancer cells, finely minced and suspended in RPMI-1640 medium containing10% FCS (HyClone) to obtain a single cell suspension. Adhesive cellsadhered to a plastic Petri dish were removed from the cell suspensionand non-adhesive cells were used as NK cells.

The NK cells were suspended in RPMI-1640 medium containing 10% FCS at aconcentration of 6×10⁶/ml. 100 μl/well of the suspension was seeded intoa microtiter plate.

Target cells were prepared as follows. 3700 kBq of ⁵¹Cr (Amersham) wasadded to 1×10⁶ YAC-1 cells (Dainippon Pharmaceutical). The cells werecultured at 37° C. for 1 hour for labeling. The labeled cells werewashed twice and then suspended in RPMI-1640 medium containing 10% FCSat a concentration of 2×10⁵ cells/ml. 100 μl of the thus prepared targetcells were overlaid onto each well of the plate into which NK cells hadbeen added. The plate was incubated at 37° C. for 5 hours in a 5% CO₂incubator. After incubation, the plate was centrifuged at 1500 rpm for 5minutes. 100 μl of the supernatant was collected in a gamma countertube. The radioactivity (experimental value) was measured using an autogamma counter.

100 μl of the culture medium was added to the reaction system in placeof the NK cells and the radioactivity was measured in order to measurethe radioactivity spontaneously released from the target cells (controlvalue).

Furthermore, the radioactivity released into the supernatant when 1%Triton (Nacalai Tesque) was added to the reaction system to lyse the₅₁Cr-labeled YAC-1 cells was measured in order to measure the totalradioactivity contained in the reaction system (total radioactivityvalue).

The cytotoxicity (%) specifically mediated by the NK cells can bedetermined according to the following equation.

Cytotoxicity (%)=[(experimental value−control value)/(totalradioactivity value−control value)]×100

The results are shown in Table 5. Activation of NK cells was observed inmice administered with cyclopentenone, GM or dipropionylcyclopentenone.The administration of these compounds increased the immunologicalprotection mechanism in a living body and cytotoxic activity againstcancer cells. In addition, optical isomers of the respective compoundsand salts thereof exhibited similar activities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

TABLE 5 Dose Cytotoxicity Test substance (mg/kg) (%) Control 3.5Cyclopentenone 10 15.5 GM 30 19.0 Dipropionyl- 10 13.5 cyclopentenone

EXAMPLE 9

Lewis rats (male, 9 weeks old, weighing about 250 g) were purchased fromSeac Yoshitomi.

Rats were fasted from 18 hours before the start of the experiments.Dipropionylcyclopentenone, dihexanoylcyclopentenone,di-2-hexenoylcyclopentenone, diisobutyrylcyclopentenone ordibenzoylcyclopentenone dissolved in olive oil (Nacalai Tesque) wasorally administered to 4 rats per group at 1 or 10 mg/10 ml/kg.

100 μl of 1% λ-carrageenan (Wako Pure Chemical Industries) suspension insaline (Otsuka Pharmaceutical) was injected into the sole of right pawof a rat 0.5 hour after the administration of test substance to inducepedal edema. The volume of the right foot of the rat was measured usingan instrument for measuring pedal volume (Ugo Basile) 3 hours after theinjection with carrageenan. The measurements were expressed as the rateof increase calculated based on the volume of the right foot of each ratmeasured before the administration with carrageenan.

The results are shown in FIG. 5. FIG. 5 illustrate the relationshipbetween the amount of cyclopentenone derivative administered and therate of increase in edema in foot. The horizontal axis represents thedose (mg/ml) and the vertical axis represents the rate of increase (%).

Tendency to suppress the edema in the foot was observed whendipropionylcyclopentenone was administered at 1 mg/kg. Administration at10 mg/kg resulted in significant suppression. Administration ofdihexanoylcyclopentenone or di-2-hexenoylcyclopentenone at 1 or 10 mg/kgresulted in significant suppression. Tendency of suppression wasobserved when diisobutyrylcyclopentenone or dibenzoylcyclopentenone wasadministered at 1 mg/kg. Administration at 10 mg/kg resulted insignificant suppression. In addition, optical isomers of thecyclopentenone derivatives and salts thereof exhibited similaractivities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

In FIG. 5, marks * and ** represent significant differences of p<0.05and p<0.01 as compared with the control group, respectively.

EXAMPLE 10

C57BL/6 mice (male, 7 weeks old, weighing about 25 g) were purchasedfrom Japan SLC and pre-bred for 1 week before using in experiments.

Sheep erythrocytes (Shimizu Laboratory Supplies) as raising antigenswere washed three times with saline (Otsuka Pharmaceutical) andsuspended in saline at a concentration of 1×10⁹ cells/ml. 200 μl of thesuspension was intraperitoneally injected into the mouse forsensitization with the antigen. 40 μl of similarly prepared antigen wasinjected to the sole of right paw 5 days after the sensitization forantigenic induction to induce pedal edema.

Dipropionylcyclopentenone dissolved in saline at a varying concentrationwas intraperitoneally or orally administered to 5 mice per group once aday for 3 days from the day of sensitization with the antigen. Thevolume of the right foot of the mouse was measured using an instrumentfor measuring pedal volume (Ugo Basile) 2 days after the antigenicinduction and used as an index of DTH. The measurements were expressedas the rate of increase calculated according to the following equationbased on the volume of the right foot of each mouse measured before theantigenic induction.

Rate of increase=(volume of right foot after antigenic induction−volumeof right foot before antigenic induction)/volume of right foot beforeantigenic induction

The results are shown in FIG. 6. In FIG. 6, the vertical axis representsthe rate of increase in the volume of foot increased by the antigenicinduction. Tendency to suppress the edema in the foot was observed whendipropionylcyclopentenone was intraperitoneally administered at 1 mg/kg.Intraperitoneal administration at 10 mg/kg or oral administration at 30mg/kg resulted in significant suppression. In addition, optical isomersof the cyclopentenone derivatives and salts thereof exhibited similaractivities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

In FIG. 6, marks * and ** represent significant differences of p<0.05and p<0.01 as compared with the control group, respectively.

EXAMPLE 11

(1) A spleen was taken out from ddY mouse (male, 7 weeks old, purchasedfrom Japan SLC), finely minced and suspended in RPMI-1640 mediumcontaining 10% FCS (HyClone) to obtain a single cell suspension. Thecell suspension was seeded into a plastic Petri dish and cultured at 37°C. for 2 hours in a CO₂ incubator to remove adhesive cells adhered tothe dish. Non-adhesive cells were used as spleen lymphocytes. 200 μl ofsuspension of the spleen lymphocytes at a cell concentration of 2×10⁶/mlwas seeded into each well of a 96-well microtiter plate.Dipropionylcyclopentenone at a varying concentration was added to eachwell other than the control well. Furthermore, 5 μg of Con A (NacalaiTesque) was added to each well. The plate was incubated at 37° C. for 1day in a CO₂ incubator. After incubation, 1 μCi of ³H-thymidine wasadded to each well. After culturing for additional 1 day, the uptakeinto cells was measured using a liquid scintillation counter.

The results are shown in FIG. 7. FIG. 7 illustrates the relationshipbetween the concentration of dipropionylcyclopentenone and the amount of³H-thymidine uptake. The horizontal axis representsdipropionylcyclopentenone concentration (μM) and the vertical axisrepresents the ³H-thymidine uptake (CPM). The open bar represents the³H-thymidine uptake without stimulation. The shaded bar represents the³H-thymidine uptake when cells were stimulated with Con A. As seen fromFIG. 7, dipropionylcyclopentenone exhibited an inhibitory activityagainst the proliferation of mouse lymphocytes stimulated with a mitogenin a dose-dependent manner. The proliferation was almost completelyinhibited at a concentration of 10 μM. In addition, optical isomers ofthe cyclopentenone derivatives and salts thereof exhibited similaractivities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

(2) Spleens were taken out from a BALB/c mouse (male, 6 weeks old,purchased from Japan SLC) and a C57BL/6 mouse (male, 6 weeks old,purchased from Japan SLC) and spleen lymphocytes were obtained accordingto the method as described in Example 11-(1). The concentration of eachof the cell suspensions was adjusted to 2×10⁶ cell/ml, 100 μl portionsfrom respective suspensions were mixed together and seeded into a96-well microtiter plate. Dipropionylcyclopentenone at a varyingconcentration was added to the wells other than the control well, andthe plate was incubated at 37° C. for 4 days in a CO₂ incubator. Afterincubation, 1 μCi of ³H-thymidine was added to each well, and the platewas incubated for additional 1 day. The uptake into cells was measuredusing a liquid scintillation counter.

The results are shown in FIG. 8. FIG. 8 illustrates the relationshipbetween the concentration of dipropionylcyclopentenone and the amount of³H-thymidine uptake. The horizontal axis represents thedipropionylcyclopentenone concentration (μM) and the vertical axisrepresents ³H-thymidine uptake (CPM). The open bar represents the³H-thymidine uptake when cells from one of the lines were usedindependently. The shaded bar represents the ³H-thymidine uptake whencells from both of the lines were mixed together. As seen from FIG. 8,dipropionylcyclopentenone exhibited an inhibitory activity againstlymphocytes activated by stimulation with an alloantigen in adose-dependent manner. The reaction was almost completely inhibited at aconcentration of 1 μM. In addition, optical isomers of thecyclopentenone derivatives and salts thereof exhibited similaractivities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

EXAMPLE 12

(1) A model for endotoxin shock was constructed using CDF1 mice (8 weeksold, female, purchased from Japan SLC). Distilled water (control), or 10mg/kg of diisopropionylcyclopentenone or diisobutyrylcyclopentenone wassubcutaneously administered to a mouse. 20 μg of lipopolysaccharide(LPS; Sigma) was intraperitoneally administered to each mouse 30 minutesafter the administration. Blood was collected from the mouse 1.5 hoursafter the administration with LPS. The amount of tumor necrosis factor(TNF)-α in a serum separated from the collected blood was measured usinga commercially available ELISA kit (R&D). Each group consisted of 4mice.

The results are shown in Table 6.

TABLE 6 Dose Amount of TNF (ng/ml) Group (mg/kg) mean ± SE Control 7.53± 0.48 Diisopropionyl- 10 4.61 ± 0.57** cyclopentenone Diisobutyryl- 103.39 ± 0.33*** cyclopentenone

TNF-α production was significantly suppressed in the groups to which 10mg/kg of diisopropionylcyclopentenone or diisobutyrylcyclopentenone wasadministered as compared with the control group to which distilled waterwas administered. In Table 6, marks ** and *** represent significantdifferences of p<0.01 and p<0.001 as compared with the control group,respectively. In addition, optical isomers of the cyclopentenonederivatives and salts thereof exhibited similar activities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

(2) A model for endotoxin shock was constructed by intraperitoneallyadministering 20 μg of LPS to a CDF1 mouse (8 weeks old, female). 30 or100 mg/kg of dipropionylcyclopentenone or diisobutyrylcyclopentenone wasorally administered 30 minutes before the administration with LPS. Bloodwas collected from the mouse 1.5 hours after the administration withLPS. The amount of TNF-α in a serum separated from the collected bloodwas measured using an ELISA kit. Each group consisted of 4 mice.

The results are shown in Table 7. The oral administration ofdipropionylcyclopentenone or diisobutyrylcyclopentenone suppressed theTNF production in a dose-dependent manner as compared with the controlgroup. In addition, optical isomers of the cyclopentenone derivativesand salts thereof exhibited similar activities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

TABLE 7 Dose TNF (ng/ml) Group (mg/kg) mean ± SE Control — 5.72 ± 1.02Dipropionyl- 30 2.95 ± 0.14* cyclopentenone 100 1.26 ± 0.15**Diisobutyryl- 30 3.92 ± 0.13 cyclopentenone 100 2.48 ± 0.43* *, **p <0.05, 0.01 vs control.

EXAMPLE 13

(1) Mouse leukemia P-388 (1.1×10⁶ cells/mouse) was intraperitoneallyadministered to a 7-weeks old female DBA/2 mouse. Immediately after theadministration, a single dose of 10 mg/kg dipropionylcyclopentenone wasintraperitoneally administered. On the other hand, saline wasintraperitoneally administered to a control group in a similar manner.Survival number of mice, average days of survival and prolongation ratewere calculated for 2 groups each consisting of 8 mice.

As a result, the average days of survival were 10.1 days for the controlgroup. The average days of survival were 13.9 days for the groupadministered with dipropionylcyclopentenone. The prolongation rate wascalculated as 137.0%. Thus, a significant prolongation effect wasobserved.

Similarly, examination was carried out using a system of Meth A tumorcells: BALB/c mouse. As a result, the average days of survival were 13.4days for the control group. The average days of survival were 16.9 daysfor the group administered with dipropionylcyclopentenone. Theprolongation rate was calculated as 126.2%, indicating a significantprolongation effect.

In addition, optical isomers of the cyclopentenone derivatives and saltsthereof exhibited similar activities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

(2) Mouse solid tumor Meth A (2×10⁶ cells/mouse) was subcutaneouslyinjected into a back of a 8-weeks old female BALB/c mouse. Subsequently,dipropionylcyclopentenone was subcutaneously injected at the same sitefor successive 5 days. Saline was subcutaneously injected to a controlgroup in a similar manner. Each group consisted of 8 mice.

After 2 weeks, the cancerous tissue formed in the back of the mouse wastaken out and the weight of the tissue was measured.

The results are shown in Table 8. The average weight of cancer was 0.61g for the control group. The average weight was 0.05 g for the groupadministered with dipropionylcyclopentenone. The inhibition rate wascalculated as 92.3%. No cancerous tissue was formed in 5 out of 8 mice.In addition, optical isomers of the cyclopentenone derivatives and saltsthereof exhibited similar activities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

TABLE 8 Weight of cancer (g) Inhibition rate Group mean ± SE (%) Control0.61 ± 0.20 — Dipropionyl- 0.05 ± 0.08 92.3 cyclopentenone

(3) The carcinostatic activities of dipropionylcyclopentenone anddiisobutyrylcyclopentenone against various ascitic cancers were examinedusing two types of cancer cells, Ehrlich ascites carcinoma (EAC) andMeth A, administered intraperitoneally or orally at a varying dose. Thenumber of intraperitoneally transplanted cells and the host animals areshown in Table 9.

TABLE 9 Number of Cancer cells Mouse transplanted cells EAC ddY 1.2 ×10⁶ (female, 5 weeks old) Meth A BALB/c 2.0 × 10⁶ (female, 7 weeks old)

Dipropionylcyclopentenone or diisobutyrylcyclopentenone wasintraperitoneally or orally administered for 5 successive days from theday after the transplantation with cancer cells. Injectable distilledwater was administered to a control group in a similar manner. Averagedays of survival, prolongation rate and 30-day survival number werecalculated for groups each consisting of 8 mice.

The results are shown in Tables 10 to 13. Table 10 shows theprolongation effects when the respective test substances wereintraperitoneally administered to mice transplanted with EAC. Table 11shows the prolongation effect when the respective test substances wereintraperitoneally administered to mice transplanted with Meth A. Table12 shows the prolongation effects when the respective test substanceswere orally administered to mice transplanted with EAC. Table 13 showsthe prolongation effect when the respective test substances were orallyadministered to mice transplanted with Meth A.

Dipropionylcyclopentenone and diisobutyrylcyclopentenone exhibitedexcellent prolongation effects in the mice transplanted with EAC. Inparticular, 30-day survival mice were observed in both of the groups ofintraperitoneal administration. Dipropionylcyclopentenone exhibitedstrong effects even if it was orally administered.

Furthermore, prolongation effect was observed for the group treated byintraperitoneal administration among the mice transplanted with Meth A.30-day survival mice were observed in the group of oral administrationwith dipropionylcyclopentenone.

In addition, optical isomers of the cyclopentenone and salts thereofexhibited similar activities.

Furthermore, other cyclopentenone derivatives or optical isomersthereof, or salts thereof exhibited similar activities.

TABLE 10 Average days Prolonga- 30-day Dose of survival tion ratesurvival Group (mg/kg) (days) (%) number Control 12.5 100 0 Dipropionyl-10 28.1 >225 6 cyclopentenone Diisobutyryl- 30 25.3 >202 3cyclopentenone 10 17.0 136 0

TABLE 11 Average days Prolonga- 30-day Dose of survival tion ratesurvival Group (mg/kg) (days) (%) number Control 11.8 100 0 Dipropionyl-10 20.5 174 0 cyclopentenone Diisobutyryl- 10 15.5 132 0 cyclopentenone

TABLE 12 Average days Prolonga- 30-day Dose of survival tion ratesurvival Group (mg/kg) (days) (%) number Control 12.5 100 0 Dipropionyl-30 24.1 >193 4 cyclopentenone Diisobutyryl- 100 20.9 167 0cyclopentenone 30 15.1 121 0

TABLE 13 Average days Prolonga- 30-day Dose of survival tion ratesurvival Group (mg/kg) (days) (%) number Control 11.8 100 0 Dipropionyl-30 17.3 >147 2 cyclopentenone

EXAMPLE 14

Injectable Preparation

(1) Diisobutyrylcyclopentenone, dipropionylcyclopentenone ordimethoxycyclopentenone was added to saline at a concentration of 1% toprepare injectable preparations.

(2) Dimethylfumarylcyclopentenone or dimethylmaleylcyclopentenone andglycyrrhizic acid was added to saline at concentrations of 0.5% and0.1%, respectively, to prepare injectable preparations.

EXAMPLE 15

Tablet

(1) Tablets each containing 100 mg of diisobutyrylcyclopentenone ordipropionylcyclopentenone and a suitable amount of crystallite cellulosewere prepared and sugar-coated.

(2) Tablets each containing 0.1 mg of dimethylfumarylcyclopentenone, 10mg of glycyrrhizic acid dipotassium salt and a suitable amount ofcrystallite cellulose were prepared and sugar-coated.

As described above, the present invention provides cyclopentenonederivatives or optical isomers thereof, or salts thereof havingphysiological activities such as an immunoregulatory activity, ananti-inflammatory activity, an activity of inhibiting tumor necrosisfactor production and an antifungal activity. Pharmaceuticalcompositions containing these compounds as their active ingredients areuseful for maintaining homeostasis in a living body as pharmaceuticalcompositions for treating or preventing a disease that requiresimmunoregulation for its treatment or prevention, a disease thatrequires suppression of inflammation for its treatment or prevention, adisease that requires inhibition of tumor necrosis factor production forits treatment or prevention, a disease that requires growth inhibitionof pathological microorganism for its treatment or prevention and thelike.

What is claimed is:
 1. A method for treating or preventing a diseasethat requires immunoregulation for its treatment or prevention, adisease that requires suppression of inflammation for its treatment orprevention, a disease that requires inhibition of tumor necrosis factorproduction for its treatment or prevention, a disease that requiresinhibition of a fungus for its treatment or prevention, a disease thatrequires inhibition of cell adhesion for its treatment or prevention, adisease that requires activation of NK cells for its treatment orprevention or a disease that requires induction of heat shock proteinfor its treatment or prevention, the method comprising administering toa subject in need thereof a pharmaceutical composition which contains asan active ingredient, in an amount sufficient therefor, of at least onecompound of formula (I):

 wherein R₁ and R₂ may be identical or different from each other, andare hydrogen, an aliphatic group, an aromatic group or an aromaticaliphatic group, or an optical isomer or salt thereof.
 2. The methodaccording to claim 1, wherein R₁ and R₂ are identical or different fromeach other, and are hydrogen, a linear or branched C1-30 alkyl group, alinear or branched C2-30 alkenyl group, a C6-10 aryl group or a C1-30alkyl C6-10 aryl group, optionally substituted with at least onesubstituent selected from the group consisting of a C1-30 alkyl group, aC1-4 alkoxy group, a C2-5 alkoxycarbonyl group, an amino group, a nitrogroup, an oxo group, a hydroxyl group, a thiol group, a sulfate groupand a halogen.
 3. The method according to claim 1, wherein thecyclopentenone derivative of formula (I) is diacetylcyclopentenone,dipropionylcyclopentenone, dibutyrylcyclopentenone,diisobutyrylcyclopentenone, divalerylcyclopentenone,dihexanoylcyclopentenone, dioctanoylcyclopentenone,didecanoylcyclopentenone, dimyristoylcyclopentenone,dimethoxyacetylcyclopentenone, dimethylfumarylcyclopentenone,dimethylmaleylcyclopentenone, di-2-hexenoylcyclopentenone,di-3-octenoylcyclopentenone or dibenzoylcyclopentenone.
 4. The methodaccording to claim 1, wherein said sufficient amount is animmunoregulatory-effective amount, a tumor necrosis factor productioninhibiting-effective amount, an anti-fungal effective amount, a celladhesion inhibiting-effective amount, an NK cell activating-effectiveamount, or a heat shock protein-inducing amount.